Elevated levels of Secreted-Frizzled-Related-Protein 1 contribute to Alzheimer’s disease pathogenesis

Abstract

The deposition of aggregated amyloid-β peptides derived from the pro-amyloidogenic processing of the amyloid precurson protein (APP) into characteristic amyloid plaques (APs) is distinctive to Alzheimer’s disease (AD). Alternative APP processing via the metalloprotease ADAM10 prevents amyloid-β formation. We tested whether downregulation of ADAM10 activity by its secreted endogenous inhibitor secreted-frizzled-related protein 1 (SFRP1) is a common trait of sporadic AD. We demonstrate that SFRP1 is significantly increased in the brain and cerebrospinal fluid of patients with AD, accumulates in APs and binds to amyloid-β, hindering amyloid-β protofibril formation. Sfrp1 overexpression in an AD-like mouse model anticipates the appearance of APs and dystrophic neurites, whereas its genetic inactivation or the infusion of α-SFRP1-neutralizing antibodies favors non-amyloidogenic APP processing. Decreased Sfrp1 function lowers AP accumulation, improves AD-related histopathological traits and prevents long-term potentiation loss and cognitive deficits. Our study unveils SFRP1 as a crucial player in AD pathogenesis and a promising AD therapeutic target.

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Fig. 1: SFRP1 is upregulated in human samples from patients with AD, correlating with pro-amyloidogenic APP processing.
Fig. 2: SFRP1 binds to amyloid-β, hindering the formation of amyloid-β protofibrils.
Fig. 3: Progressive upregulation of brain Sfrp1 correlates with the age of APP;PS1 mice brains, and its forced expression accelerates the appearance of APs.
Fig. 4: Sfrp1 is expressed by glial cells in APP;PS1 mice, and its forced brain expression accelerates the appearance of APs.
Fig. 5: Sfrp1 inactivation prevents the appearance of pathological traits in APP;PS1 mice.
Fig. 6: Sfrp1 inactivation in APP;PS1 mice shifts APP processing towards the non-amyloidogenic pathway and prevents cognitive decline.
Fig. 7: Antibody-mediated neutralization of Sfrp1 activity counteracts the appearance of AD pathogenic traits in mice.

Data availability

The data that support the findings of this study are available from the corresponding authors upon request.

References

  1. 1.

    Selkoe, D. J. & Hardy, J. The amyloid hypothesis of Alzheimer’s disease at 25 years. EMBO Mol. Med. 8, 595–608 (2016).

  2. 2.

    Masters, C. L. et al. Alzheimer’s disease. Nat. Rev. Dis. Prim. 1, 15056 (2015).

  3. 3.

    Cummings, J., Lee, G., Ritter, A. & Zhong, K. Alzheimer’s disease drug development pipeline: 2018. Alzheimers Dement. (NY) 4, 195–214 (2018).

  4. 4.

    Benilova, I., Karran, E. & De Strooper, B. The toxic Abeta oligomer and Alzheimer’s disease: an emperor in need of clothes. Nat. Neurosci. 15, 349–357 (2012).

  5. 5.

    Haass, C. & Selkoe, D. J. Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer’s amyloid beta-peptide. Nat. Rev. Mol. Cell Biol. 8, 101–112 (2007).

  6. 6.

    Fowler, S. W. et al. Genetic modulation of soluble abeta rescues cognitive and synaptic impairment in a mouse model of alzheimer’s disease. J. Neurosci. 34, 7871–7885 (2014).

  7. 7.

    Sevigny, J. et al. The antibody aducanumab reduces Abeta plaques in Alzheimer’s disease. Nature 537, 50–56 (2016).

  8. 8.

    Jorissen, E. et al. The disintegrin/metalloproteinase ADAM10 is essential for the establishment of the brain cortex. J. Neurosci. 30, 4833–4844 (2010).

  9. 9.

    Lichtenthaler, S. F. Alpha-secretase in Alzheimer’s disease: molecular identity, regulation and therapeutic potential. J. Neurochem. 116, 10–21 (2011).

  10. 10.

    Suh, J. et al. ADAM10 missense mutations potentiate beta-amyloid accumulation by impairing prodomain chaperone function. Neuron 80, 385–401 (2013).

  11. 11.

    Kim, M. et al. Potential late-onset Alzheimer’s disease-associated mutations in the ADAM10 gene attenuate alfa-secretase activity. Hum. Mol. Genet 18, 3987–3996 (2009).

  12. 12.

    Esteve, P. et al. Secreted frizzled-related proteins are required for Wnt/beta-catenin signalling activation in the vertebrate optic cup. Development 138, 4179–4184 (2011).

  13. 13.

    Bovolenta, P., Esteve, P., Ruiz, J. M., Cisneros, E. & Lopez-Rios, J. Beyond Wnt inhibition: new functions of secreted Frizzled-related proteins in development and disease. J. Cell Sci. 121, 737–746 (2008).

  14. 14.

    Esteve, P. et al. SFRPs act as negative modulators of ADAM10 to regulate retinal neurogenesis. Nat. Neurosci. 14, 562–569 (2011).

  15. 15.

    Esteve, P., Crespo, I., Kaimakis, P., Sandonis, A. & Bovolenta, P. Sfrp1 modulates cell-signaling events underlying telencephalic patterning, growth and differentiation. Cereb. Cortex 29, 1059–1074 (2019).

  16. 16.

    Marcos, S. et al. Secreted frizzled related proteins modulate pathfinding and fasciculation of mouse retina ganglion cell axons by direct and indirect mechanisms. J. Neurosci. 35, 4729–4740 (2015).

  17. 17.

    Blalock, E. M. et al. Incipient Alzheimer’s disease: microarray correlation analyses reveal major transcriptional and tumor suppressor responses. Proc. Natl Acad. Sci. USA 101, 2173–2178 (2004).

  18. 18.

    Zipfel, W. R. et al. Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc. Natl Acad. Sci. USA 100, 7075–7080 (2003).

  19. 19.

    Simon, M. J. & Iliff, J. J. Regulation of cerebrospinal fluid (CSF) flow in neurodegenerative, neurovascular and neuroinflammatory disease. Biochim. Biophys. Acta 1862, 442–451 (2016).

  20. 20.

    Tammineni, P., Ye, X., Feng, T., Aikal, D. & Cai, Q. Impaired retrograde transport of axonal autophagosomes contributes to autophagic stress in Alzheimer’s disease neurons. eLife 6, e21776 (2017).

  21. 21.

    Biere, A. L. et al. Amyloid beta-peptide is transported on lipoproteins and albumin in human plasma. J. Biol. Chem. 271, 32916–32922 (1996).

  22. 22.

    Jan, A., Hartley, D. M. & Lashuel, H. A. Preparation and characterization of toxic Abeta aggregates for structural and functional studies in Alzheimer’s disease research. Nat. Protoc. 5, 1186–1209 (2010).

  23. 23.

    Upadhaya, A. R., Lungrin, I., Yamaguchi, H., Fandrich, M. & Thal, D. R. High-molecular weight Abeta oligomers and protofibrils are the predominant Abeta species in the native soluble protein fraction of the AD brain. J. Cell Mol. Med. 16, 287–295 (2012).

  24. 24.

    Ahmed, M. et al. Structural conversion of neurotoxic amyloid-beta(1-42) oligomers to fibrils. Nat. Struct. Mol. Biol. 17, 561–567 (2010).

  25. 25.

    Bieschke, J. et al. Small-molecule conversion of toxic oligomers to nontoxic beta-sheet-rich amyloid fibrils. Nat. Chem. Biol. 8, 93–101 (2011).

  26. 26.

    Sot, B. et al. The chaperonin CCT inhibits assembly of alpha-synuclein amyloid fibrils by a specific, conformation-dependent interaction. Sci. Rep. 7, 40859 (2017).

  27. 27.

    Jankowsky, J. L. et al. Mutant presenilins specifically elevate the levels of the 42 residue beta-amyloid peptide in vivo: evidence for augmentation of a 42-specific gamma secretase. Hum. Mol. Genet. 13, 159–170 (2004).

  28. 28.

    Guo, Q. et al. Amyloid precursor protein revisited: neuron-specific expression and highly stable nature of soluble derivatives. J. Biol. Chem. 287, 2437–2445 (2012).

  29. 29.

    Malinverno, M. et al. Synaptic localization and activity of ADAM10 regulate excitatory synapses through N-cadherin cleavage. J. Neurosci. 30, 16343–16355 (2010).

  30. 30.

    Guenette, S., Strecker, P. & Kins, S. APP protein family signaling at the synapse: insights from intracellular APP-binding proteins. Front. Mol. Neurosci. 10, 87 (2017).

  31. 31.

    Gowrishankar, S. et al. Massive accumulation of luminal protease-deficient axonal lysosomes at Alzheimer’s disease amyloid plaques. Proc. Natl Acad. Sci. USA 112, E3699–E3708 (2015).

  32. 32.

    Ferrer, I. Defining Alzheimer as a common age-related neurodegenerative process not inevitably leading to dementia. Prog. Neurobiol. 97, 38–51 (2012).

  33. 33.

    Satoh, W., Gotoh, T., Tsunematsu, Y., Aizawa, S. & Shimono, A. Sfrp1 and Sfrp2 regulate anteroposterior axis elongation and somite segmentation during mouse embryogenesis. Development 133, 989–999 (2006).

  34. 34.

    Condello, C., Yuan, P., Schain, A. & Grutzendler, J. Microglia constitute a barrier that prevents neurotoxic protofibrillar Abeta42 hotspots around plaques. Nat. Commun. 6, 6176 (2015).

  35. 35.

    Colombo, A. et al. Constitutive alpha- and beta-secretase cleavages of the amyloid precursor protein are partially coupled in neurons, but not in frequently used cell lines. Neurobiol. Dis. 49, 137–147 (2013).

  36. 36.

    Esteve, P. & Bovolenta, P. The advantages and disadvantages of sfrp1 and sfrp2 expression in pathological events. Tohoku J. Exp. Med. 221, 11–17 (2010).

  37. 37.

    Epis, R. et al. Blocking ADAM10 synaptic trafficking generates a model of sporadic Alzheimer’s disease. Brain 133, 3323–3335 (2010).

  38. 38.

    Serneels, L. et al. gamma-Secretase heterogeneity in the Aph1 subunit: relevance for Alzheimer’s disease. Science 324, 639–642 (2009).

  39. 39.

    Wang, Y. et al. Lessons from anti-amyloid-beta immunotherapies in Alzheimer disease: aiming at a moving target. Neurodegener. Dis. 17, 242–250 (2017).

  40. 40.

    Li, Q. et al. Metaplasticity mechanisms restore plasticity and associativity in an animal model of Alzheimer’s disease. Proc. Natl Acad. Sci. USA 114, 5527–5532 (2017).

  41. 41.

    Selkoe, D. J. Soluble oligomers of the amyloid beta-protein impair synaptic plasticity and behavior. Behav. Brain Res. 192, 106–113 (2008).

  42. 42.

    Song, W. M. & Colonna, M. The identity and function of microglia in neurodegeneration. Nat. Immunol. 19, 1048–1058 (2018).

  43. 43.

    Kuhn, P. H. et al. Systematic substrate identification indicates a central role for the metalloprotease ADAM10 in axon targeting and synapse function. eLife 5, e12748 (2016).

  44. 44.

    Saftig, P. & Lichtenthaler, S. F. The alpha secretase ADAM10: A metalloprotease with multiple functions in the brain. Prog. Neurobiol. 135, 1–20 (2015).

  45. 45.

    Godyn, J., Jonczyk, J., Panek, D. & Malawska, B. Therapeutic strategies for Alzheimer’s disease in clinical trials. Pharm. Rep. 68, 127–138 (2016).

  46. 46.

    St-Amour, I., Cicchetti, F. & Calon, F. Immunotherapies in Alzheimer’s disease: too much, too little, too late or off-target? Acta Neuropathol. 131, 481–504 (2016).

  47. 47.

    Boada, M. et al. Efficacy and safety of plasma exchange with 5% albumin to modify cerebrospinal fluid and plasma amyloid-beta concentrations and cognition outcomes in Alzheimer’s disease patients: a multicenter, randomized, controlled clinical trial. J. Alzheimers Dis. 56, 129–143 (2017).

  48. 48.

    Endres, K. & Fahrenholz, F. Upregulation of the alpha-secretase ADAM10–risk or reason for hope? FEBS J. 277, 1585–1596 (2010).

  49. 49.

    Vojdani, A. & Vojdani, E. Amyloid-beta 1-42 cross-reactive antibody prevalent in human sera may contribute to intraneuronal deposition of A-beta-P-42. Int J. Alzheimers Dis. 2018, 1672568 (2018).

  50. 50.

    Barrachina, M., Castano, E. & Ferrer, I. TaqMan PCR assay in the control of RNA normalization in human post-mortem brain tissue. Neurochem Int 49, 276–284 (2006).

  51. 51.

    Agostini, F. et al. Prion protein accumulation in lipid rafts of mouse aging brain. PLoS One 8, e74244 (2013).

  52. 52.

    Sherman, M. A. & Lesne, S. E. Detecting abeta*56 oligomers in brain tissues. Methods Mol. Biol. 670, 45–56 (2011).

  53. 53.

    Biesiadecki, B. J. & Jin, J. P. A high-throughput solid-phase microplate protein-binding assay to investigate interactions between myofilament proteins. J. Biomed. Biotechnol. 2011, 421701 (2011).

  54. 54.

    Uren, A. et al. Secreted frizzled-related protein-1 binds directly to Wingless and is a biphasic modulator of Wnt signaling. J. Biol. Chem. 275, 4374–4382 (2000).

  55. 55.

    Zhong, X. et al. Regulation of secreted Frizzled-related protein-1 by heparin. J. Biol. Chem. 282, 20523–20533 (2007).

  56. 56.

    Kleinberger, G. et al. TREM2 mutations implicated in neurodegeneration impair cell surface transport and phagocytosis. Sci. Transl. Med. 6, 243ra286 (2014).

  57. 57.

    Faul, F., Erdfelder, E., Buchner, A. & Lang, A. G. Statistical power analyses using G*Power 3.1: tests for correlation and regression analyses. Behav. Res Methods 41, 1149–1160 (2009).

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Acknowledgements

We are in debt to I. Torres-Aleman, Instituto Cajal-CSIC, for donating a breeding pair of APP;PS1 mice and to J. Avila, C. Dotti, M.L. Toribio, S. Knafo and E. Palomer (CBMSO) for their advice during the course of this study. We also acknowledge the generosity of M.L. de Ceballos, Instituto Cajal-CSIC, and M. Llorens, CBMSO, for sharing some tissue samples and C. Bovolenta (MolMed) for advice on lentiviral production. We thank O. Lancho and M.L. Toribio for the Sfrp1 lentiviral construct and M. Guerra of the CBMSO EM facilities for help with TEM. We wish to thank C. Dotti, J. Garcia de Yébenes, S.R. de Cordoba (CIB-CSIC), C. Bovolenta and M. Nieto (CNB, CSIC) for critical reading the manuscript. This work was supported by grants from the Spanish MINECO (nos. BFU2013-43213-P and BFU2016-75412-R with FEDER support), the Fundación Tatiana Perez de Guzman el Bueno and CIBERER with grants to P.B. and P.E., and grant no. FIS PI11/3035 to A.L. J.R.C. (grant no. BES-2011-047189), M.I.M. (no. BES-2014-068797) and G.P. (no. BES-2017-080318) were supported by FPI fellowships from the MINECO. We also acknowledge a CBMSO Institutional grant from the Fundación Ramon Areces.

Author information

P.E. and P.B. set up the research concept, designed the experiments and wrote the manuscript. P.E., J.R.C., M.I.M., I.C., A.S., M.J.M.B. and G.P. carried out the experiments except those in Figs. 1b,d and 2m–r and Supplementary Fig. 2, which were performed by P.G.E. and E.A. under the supervision of I.F. I.M. and M.D. produced the monoclonal antibodies and contributed to the set-up of the ELISA assays. J.D. performed LTP analysis under the supervision of J.A.E. P.E., J.R.C., M.I.M., I.C., G.P. and P.B. interpreted the data. J.R.C., P.E. and P.B. assembled the figures. I.F. and M.T.H. provided invaluable conceptual advice and J.M.V. contributed to TEM studies and interpretation. I.F. and A.R. provided human tissue samples and M.T.H., A.L., J.F., D.A. and E.G.T. provided CSF and other samples.

Correspondence to Pilar Esteve or Paola Bovolenta.

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Competing interests

The following authors are named in a patent application: Paola Bovolenta, Pilar Esteve, Mercedes Dominguez, Inmaculada Moreno, María Inés Mateo, Javier Rueda Carrasco, María Jesús Martin-Bermejo. Patent: ‘‘Therapeutic Target And Monoclonal Antibodies Against It For The Diagnosis And Treatment Of Alzheimer’s Disease.’’ Application number: EP19382105.5. Status of application: pending.

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Peer review information: Nature Neuroscience thanks Qian Cai, Giulio Taglialatela, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Integrated supplementary information

Supplementary Figure 1 Characterization of anti-SFRP1 monoclonal antibodies.

A) Representative ELISA standard curve showing the specificity of the assay that recognizes recombinant human SFRP1 B) but not the highly related human SFRP2. Experiments were independently repeated 10 times, obtaining with similar results C) Western blot analysis showing the specificity of mAb 10.5.6. Lane 1: human recombinant SFRP1, lane 2: human tear (a fluid enriched in SFRP1); lane 3: extract from E12.5 wild type embryonic telencephalon; lane 4 extract from the telencephalon of Sfrp1-/- E12.5 embryos. Note how the mAb recognizes both human and mouse tissue. D, E) Frontal cryostat sections of the telencephalon from wt and Sfrp1-/- E12.5 embryos immunostained with the 10.5.6 mAb. Sections were counterstained with Hoechst. Note specific staining in the neuroepithelium of WT (arrow in D) absent in the Sfrp1-/- embryos, where there is only unspecific signal in the ectoderm (open arrowhead in E). Experiments in C-F were independently repeated 4 times, obtaining with similar results. Scale bar: 100 μm.

Supplementary Figure 2 Amyloidogenic APP is favored in brain homogenates from AD patients without major changes in the expression of APP, ADAM10, and BACE1 in AD human cortical samples.

A-D) Western blot analysis of extracts from control (n=3) and AD patients at intermediate (III-IV; n=3) and advanced stages (V-VI; n=3) of the disease probed with the antibodies indicated at the bottom of each one of the images to detect the following APP proteolytical products: sAPPα (A), total APP (B), sAPPβ (C) and CTFα/β fragments (D). E) Quantifications of the immunoreactive bands normalized to those of the respective full lengths APP levels. Data are means ± SEM analyzed with two-sided Student-t test. No significant differences were observed although there was a clear tendency towards an increased production of amyloidogenic sAPPβ fragment in AD patients and a reduced in non-amyloidogenic sAPPα peptide. F) The graphs show fold enrichment of APP, ADAM10 and BACE1 mRNA levels in the entorhinal (EC) and frontal (FC) cortex from control (n=5 and n=11, respectively) and AD I-II (n=5 for both EC and FC), III-IV (n=4 and n=9, respectively) and V-VI samples (n=5 and n=19 respectively; see Supplementary Table 1, for sample description). Values were determined by Taqman PCR assays, normalized against the levels of HPRT and AARS housekeeping genes (see Supplementary Table 3 for primer sequence). Note that there is only a significant increase of APP (P=0.0325) and ADAM10 (P=0.0094) levels in the frontal cortex at late stages of the disease when compared to control samples. Data are means ± SEM analyzed with Kruskal-Wallis followed by post hoc Mann-Whitney test. Differences with respect to controls were considered statistically significant at p-value: * p≤0.05; ** p≤0.01; ns, not statistically significant. All experiments were repeated at least three times, obtaining similar results.

Supplementary Figure 3 Extended data from Fig. 1. SFRP1 specifically localizes to APs, blood vessels and choroid plexus.

A-D; F-H) Paraffin sections of the frontal cortex from BBV-VI AD patients (A-C; F-H) and age matched controls (D) coimmunostained for SFRP1 and Aβ, ThioS or probed with secondary antibodies only, as indicated in the panels. Note that SFRP1 specifically localizes to blood vessels identified by the autofluorescence of elastin16 (A). B,C) No signal was detected with secondary antibodies, supporting the specificity of SFRP1 localization. D) No detectable signal was found in control sections. E) Western blot analysis of SFRP1 presence in choroid plexus extracts from AD (n=5) patients and cognitive normal controls (n=4), normalized to α-Tubulin. Data are means ± SEM and were analyzed with two-sided Student-t test, finding no significant differences. F-H) SFRP1 specifically accumulates in the core of Aβ+ and ThioS+ APs (white arrowheads) and their surroundings (yellow arrows in F,G) and forms aggregates that are poorly ThioS+ (arrowhead in H). These experiments were repeated 4 times with similar results. Scale bar: 50 μm A-C; 100 μm D-G.

Supplementary Figure 4 Extended data from Fig. 2. SFRP1 interacts with Aβ and reduces the time-dependent formation of the less harmful Aβ1-42 protofibrils.

A, B) Western blot analysis of Aβ or SFRP1 interaction with BSA, Heparin, SFRP1 or Aβ after microplate-based solid-phase protein-protein binding assay as indicated in the panels. Note that Aβ seems to bind more efficiently to SFRP1 than to BSA. C) Examples of transmission electron microscopic images showing the conformation of Aβ1-42 and SFRP1 alone or in combination, after incubation at 37°C for the indicated times. Note that SFRP1 favors the oligomeric (red arrows) vs proto-fibrillary (black arrows) form of Aβ1-42, which are instead prevalent when Aβ1-42 peptides are incubated alone. SFRP1 alone also tends to form aggregates. D) Dot blot analysis of samples with different concentrations of Aβ1-42 and SFRP1 alone or in combination at 0 or 24 hr of incubation at 37°C. Membranes were probed to detect the presence and relative abundance of Aβ1-42 fibril/protofibril forms (OC antibody) and low molecular weight amyloid oligomers (A11 conformation antibody) as well as control anti- SFRP1 and Aβ1-42 (6E10) antibodies. Note that Aβ1-42 fibril/protofibril forms increases with time (compare time 0h with 24h) but their relative abundance is decreased in the presence of SFRP1. Note that the A11 antibody recognizes also SFRP1. Scale bar 80 nm. These experiments were repeated three times obtaining similar results.

Supplementary Figure 5 Specific and selective distribution of Sfrp1 in wt and APP;PS1 mouse cortex.

A) Western blot analysis of Sfrp1 expression in cortical extracts from wt, APP;PS1 and APP;PS1;Sfrp1-/- animals at the ages indicated in the panels. Note that SFRP1 expression increases with the progression of the disease/age in APP;PS1 mice, as shown in the graph. B-E) Frontal cryostat sections of the cortex from 4.5-months-old wt, APP;PS1 and APP;PS1;Sfrp1-/- mice co-immunostained for Sfrp1 and GFAP or Iba1 or laminin (LN), as indicated in the panels. Note that Sfrp1 localizes to the walls of the lateral ventricle overlapping with some GFAP+ and Iba1+ cells. It is also found in LN+ blood vessels. Sfrp1 staining in blood vessels is not detected in mice lacking Sfrp1 (E). F-I) Cryostat sections from the cortex of 9-months-old APP;PS1 and APP;PS1;Sfrp1-/- mice immunostained with 10.5.6 mAb or with a rabbit polyclonal (Abcam ab4193) against Sfrp1 and counterstained with ThioS, as indicated in the panels. Sfrp1 localizes to ThioS+ plaques in APP;PS1 mice with both antibodies. No Sfrp1+ signal was observed in the few ThioS+ APs present in APP;PS1;Sfrp1-/- mice strongly supporting the specificity of the signal in the AP. Scale bar: 200 μm B,C, F-I; 100 μm D, E. These experiments were repeated 3 times with similar results.

Supplementary Figure 6 Extended data from Fig. 3. Uncropped Western Blot images.

A-C) Uncropped Western Blot images displayed in Fig. 3l, m, and n, respectively.

Supplementary Figure 7 Extended data from Fig. 4. Forced expression of Sfrp1, which is endogenously produced by astrocytes and microglial cells, accelerates the appearance of APs.

A-J) Frontal sections of the cortex from 3 months-old APP;PS1 mice transduced one month earlier with lentiviral particles (LV) expressing GFP (n=6) or Sfrp1-IRES-GFP(n=9) as indicated in the panel. Sections in A, B show anti-GFP immunostaining reflecting the LV infection close to the lateral ventricle (v) and extending dorsally to the cortex. Sections in C-J were co-immunostained for Aβ and GFAP or CD45, or stained with ThioS and immunostained for p-Tau, as indicated in the panels. Note that transduction with Sfrp1-LV promotes the formation of AP and increases the associated reactive gliosis. K) The graph shows the quantification of the number of APs found in the cortex of infected mice. Data are means ± SEM analyzed with two-sided Student-t test; P=0.00012; ***p≤0.0001. L-P) Cryostat sections of the cortex from 4.5-months-old APP;PS1;Sfrp1-/- mice coimmunostained with antibodies against nuclear β-Gal and GFAP, NeuN or Iba1, or stained with ThioS, as indicated in the panels. Note that the nuclear β-Gal, reflecting Sfrp1 expression, localizes to GFAP+ and Iba1+ cells, but no to NeuN+ cells. Note also the strong β-Gal signal in cells close to ThioS+ plaques. These experiments were repeated 4 times obtaining similar results. Scale bar: 200 μm A-D; 100 μm E-J and L-O; P, 50 μm.

Supplementary Figure 8 Extended data from Fig. 5. Sfrp1 inactivation critically delays the appearance of amyloid plaques in APP;PS1 mice.

A) Quantification of the size distribution of Aβ+ APs illustrated in Fig. 5a–f. Note the significant decrease in the size of Aβ+ hotspots in Sfrp1 absence. Data are means ± SEM analyzed with two-sided Student-t test. B-E, G,H) Frontal cryostat sections of the cortex from 4.5, 9 and 20 months-old APP;PS1 and APP;PS1;Sfrp1-/- mice stained with ThioS as indicated in the panels. F) Quantification of the number of ThioS+ APs in 4.5 (n=6, both genotypes), 9 (n=4, both genotypes) and 20 (n=3, both genotypes) months-old APP;PS1 and APP;PS1;Sfrp1-/- mice. I-M) Frontal sections of the cortex from a different set of 4.5 and 9 months-old APP;PS1 and APP;PS1;Sfrp1-/- mice stained with antibodies against Aβ40-42. K) Quantification of the number of Aβ40-42+ APs in 4.5 (n=6, both genotypes), 9 (n=4, both genotypes) APP;PS1 and APP;PS1;Sfrp1-/- mice. *p≤0.05; **p≤0.01; ***p≤0.001. Scale bar: 100 μm.

Supplementary Figure 9 Extended data from Fig. 5. Activation of glial cells is reduced in APP;PS1;Sfrp1-/- mice.

A-H) Frontal sections of the cortex from 9 months-old APP;PS1 and APP;PS1;Sfrp1-/- (n=3) mice immunostained with antibodies against LAMP1, Iba1 CD45 and Aβ42 or GFAP and stained with ThioS, as indicated in the panels. (I, J) Quantification of LAMP1 hotspots and Iba1, CD45 and GFAP immunoreactivity in 4.5 (n=3, for both genotypes) and 9 (n=3 or 4, for both genotypes) months-old cortex of APP;PS1 and APP;PS1;Sfrp1-/- mice. Data are means ± SEM and were analyzed with two-way ANOVA followed by Bonferroni test. *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001. Number of analyzed animals is indicated in the bars. Scale bar: 200μm.

Supplementary Figure 10 Extended data from Fig. 6. ADAM10 activity is increased in the absence of Sfrp1.

A) Uncropped nitrocellulose membrane of the IP assay stained with Ponceau and WB images displayed in Fig. 6c. B) Schematic representation of the APP molecule to indicate which are the fragments recognized by each one of the antibodies against APP used in this study. The Aβ peptide is depicted in red. C) Uncropped Western blot images shown in Fig. 6e. D) Western blot analysis of N-cadherin (Cdh2) processing in APP;PS1 (n=3) and APP;PS1;Sfrp1-/- (n=3) brains. Values were normalized to α-Tubulin levels used as a loading control. Cleaved versus full-length levels of the protein were used to determine the degree of its processing, which is significantly increased in the absence of Sfrp1, indicating a higher ADAM10 activity. Data are means ± SEM analyzed with two-sided Student-t test; P=0.011. *p≤0.05.

Supplementary Figure 11 mRNA and protein levels of APP, ADAM10, BACE1 and Axin2 are not affected by Sfrp1 inactivation in APP;PS1 mice.

A) RT-PCR analysis of App, Adam10, Axin2 and GAPDH mRNA from APP;PS1 (n=3) and APP;PS1;Sfrp1-/- (n=3) brains. Note that mRNA levels are similar in both mouse lines, as normalized with control GAPDH levels. B-D) Western blot analysis of BACE1 (B), ADAM10 (C) and Axin2 (D) protein levels in APP;PS1 and APP;PS1;Sfrp1-/- brains. Data were normalized to α-Tubulin levels used as a loading control. The data are means ± SEM analyzed with two-sided Student-t test; n.s. not statistically significant.

Supplementary Figure 12 Extended data from Fig. 6. Behavioral analysis of wt, Sfrp1-/-, APP;PS1 and APP;PS1 Sfrp1-/- mice.

A-C) The graphs depict the recognition index (A), spatial memory retention (B) and number of crossing (C) of wt (n=4) and Sfrp1-/- (n=4) 8 months-old mice subjected to the object recognition test, water maze and open field exploration, respectively. Note that the behavior of Sfrp1-/- mice was undistinguishable from that of wt mice. D, E) The graphs depict the time in quadrant (D) and number of crossing (E) of wt (n=6), APP;PS1 (n=5) and APP;PS1;Sfrp1-/- (n=6) of 8 months-old mice subjected to water maze and open field exploration, respectively. Note that APP;PS1 mice showed a poor memory, whereas APP;PS1;Sfrp1-/- mice were undistinguishable from wt. APP;PS1;Sfrp1-/- mice showed a slightly lower locomotor performance. Data are means ± SEM analyzed with two-sided Student-t test (A-C) or One-way ANOVA followed by Tukey method (D-E). *p≤0.05.

Supplementary Figure 13 Extended data from Fig. 7. mAb 10.5.6 neutralizes SFRP1 activity.

Cultured cells release into the culture medium the sAPPα peptide derived from ADAM10-mediated proteolytic processing of APP 16. Sfrp1 binds to ADAM10 and prevents APP processing thus decreasing the amount of sAPPα in the media of cultured cells 14. A) The panels show Western blot analysis of media from wt and Sfrp1-/- cultures immunoprecipitated with anti-Aβ (6E10) antibody and analyzed with the same antibody. Telencephalic neuroepithelial cells express high level of Sfrp1 mRNA 15 therefore cultured telencephalic cells from E13.5 wt embryos release undetectable levels sAPPα into the medium because the activity of ADAM10, a ubiquitously expressed protein, is inhibited. Sfrp1-/- telencephalic cultures instead release detectable sAPPα levels (compare control lanes in the two cultures) because ADAM10 is free to process APP. The addition of the 10.5.6 IgG1 against Sfrp1, but not that of an unspecific IgG of the same isotype to the medium of wt cultures enhanced sAPPα release, indicating its ability of neutralizing Sfrp1 function. No changes in the amount of sAPPα were instead observed in the Sfrp1-/- cultures. B) Quantification of the changes observed in three independent experiments performed in the same conditions as those illustrated in A. Data are means ± SEM analyzed with two-sided Student-t test; α-SFRP1 vs C P=0.00047; α-SFRP1 vs IgG1 P=0.0058., **p≤0.01; ***p≤0.001; n.s. not statistically significant.

Supplementary Figure 14 Extended data from Fig. 7. Anti-Sfrp1 antibodies enter the brain parenchyma via intravenous administration without inducing macrophage infiltration.

A-F) Frontal sections of the brain from APP;PS1 mice injected 24 hrs before in the retro-orbital sinus with biotinylated-versions of either 10.5.6 mAb or unspecific mouse IgG1 as indicated in the panel. Sections were stained with streptavidin-POD followed by tyramide amplification and immunostained for Aβ42. Note the accumulation of the 10.5.6 mAb in the brain parenchyma especially around APs and pia surface, whereas the unspecific IgG1 is poorly detected in comparable sections. G-V) Frontal sections of the brain from APP;PS1 mice treated for 2 months with IgG1 or a-Sfrp1 immunostained with anti-laminin (LN) and CD45 antibodies to visualize blood vessels and infiltrated macrophages respectively. Arrowheads point to macrophages infiltrated in the brain parenchyma whereas arrows to those found within the blood vessels. Note the poor macrophagic infiltration especially in animals treated with a-Sfrp1 IgG. These experiments were repeated three times obtaining similar results. Scale bar: 50 μm A-F; 100 μm G-V.

Supplementary Figure 15 Proposed model for Sfrp1 activity in AD.

The drawing summarizes the mechanism by which SFRP1 contributes to AD pathogenesis. SFRP1 is upregulated in activated glial cells, by still unknown mechanisms that may include general inflammatory events associated with aging, strokes etc. SFRP1 secreted into the brain parenchyma negatively regulates ADAM10-mediated APP proteolysis in neurons, favoring the pro-amyloidogenic cascade and thus the generation of more Aβ peptides and their deposition. This, in turn, upregulates inflammatory events and more Sfrp1 over-production, creating a vicious cycle that exacerbates AD pathology.

Supplementary information

Supplementary Figs. 1–15, Supplementary Methods, and Supplementary Tables 1–3.

Reporting Summary

Supplementary Video 1

SFRP1 is found in human astrocytes.

Supplementary Video 2

SFRP1 is found in human microglial cell.

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